Individual DNA sequence variations in the human genome are known to directly cause specific diseases or conditions, or to predispose certain individuals to specific diseases or conditions. Such variations also modulate the severity or progression of many diseases. Additionally, DNA sequence variations between populations. Therefore, determining DNA sequence variations in the human genome is useful for making accurate diagnoses, for finding suitable therapies, and for understanding the relationship between genome variations and environmental factors in the pathogenesis of diseases and prevalence of conditions.
There are several types of DNA sequence variations in the human genome. These variations include insertions, deletions and copy number differences of repeated sequences. The most common DNA sequence variations in the human genome, however, are single base pair substitutions. These are referred to as single nucleotide polymorphisms (SNPs) when the variant allele has a population frequency of at least 1%.
SNPs are particularly useful in studying the relationship between DNA sequence variations and human diseases and conditions because SNPs are stable, occur frequently and have lower mutation rates than other genome variations such as repeating sequences. In addition, methods for detecting SNPs are more amenable to being automated and used for large-scale studies than methods for detecting other, less common DNA sequence variations.
A number of methods have been developed which can locate or identify SNPs. These methods include dideoxy fingerprinting (ddF), fluorescently labeled ddF, denaturation fingerprinting (DnF1R and DnF2R), single-stranded conformation polymorphism analysis, denaturing gradient gel electrophoresis, heteroduplex analysis, RNase cleavage, chemical cleavage, hybridization sequencing using arrays and direct DNA sequencing.
The known methods for locating or identifying SNPs are associated with certain disadvantages. For example, some known methods do not identify the specific base changes or the precise location of these base changes within a sequence. Other known methods are not amenable to analyzing many samples simultaneously or to analyzing pooled samples. Still other known methods require different analytical conditions for the detection of each variation. Additionally, some known methods cannot be used to quantify known SNPs in genotyping assays. Further, many known methods have excessive limitations in throughput.
Thus, there is a need for a new method to determine the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide, including the presence of an SNP in the genome of a human individual. Preferably, the method could determine the presence and identity of a variation in a nucleotide sequence between a first polynucleotide and a second polynucleotide in a pooled sample. Additionally preferably, the method could determine whether two or more variations reside on the same or different alleles in an individual, and could be used to determine the frequency of occurrence of the variation in a population. Further preferably, the method could screen large numbers of samples at a time with a high degree of accuracy.